Doctoral thesis
Open access

The role of phosphatidylinositol transfer proteins Nir2 and Nir3 during phagocytosis

ContributorsKaba Lunghi, Mayis
Imprimatur date2023
Defense date2023

Phagocytosis is an essential cellular process that plays a crucial role in both innate and acquired immunity, as well as in tissue homeostasis. During phagocytosis, dynamic changes in membrane phosphoinositides accompanied by local Ca2+ elevations at the phagocytic receptorligand engagement site contribute to the remodeling of the plasma membrane and to the recruitment and activation of actin-binding proteins, which drive the formation of phagocytic cups, actin dependent invaginations of the membrane at sites of particle engulfment. Altogether, these changes are necessary for the internalization of the target and are therefore crucial for efficient phagocytosis. Nir2 (PITPNM1) and Nir3 (PITPNM2) are lipid transfer proteins with an NH2 terminus PI transfer domain (PITP). Previous studies have demonstrated their importance for sustaining plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] levels by mediating non-vesicular transfer of phosphatidic acid (PA) from the plasma membrane to the endoplasmic reticulum (ER) membrane upon agonist stimulation. PA is then converted to phosphatidylinositol (PI) at the ER membrane which is transferred back to the plasma membrane via Nir2 and Nir3 proteins to replenish PI(4,5)P2 during receptor activation. However, their role during the phagocytic process is unknown. In this thesis project, we showed that the phosphatidylinositol lipid transfer proteins Nir2 and Nir3 regulate phagocytic activity by maintaining membrane PI(4,5)P2 levels and actin contractility, leading to successful target internalization. Mouse embryonic fibroblasts (MEF) edited for Nir2 and Nir3 using CRISPR-Cas9 technology displayed decreased levels of basal plasma membrane PI(4,5)P2 and store-operated Ca2+ entry (SOCE). In Nir2/3-edited MEFs rendered phagocytic by the expression of the FcγRIIa, the decrease in the plasma membrane PI(4,5)P2 levels correlated with a reduction in phagocytosis. Moreover, in cells depleted of Nir2/3, the overall phagosomal PI(4,5)P2 levels were reduced throughout phagocytosis, but periphagosomal PI(4,5)P2 and Ca2+ elevations were still observed during particle internalization. Rescue with either of the Nir proteins restored the basal plasma membrane PI(4,5)P2 levels and the phagocytic activity but not SOCE. Nir2/3 edited cells presented elevated numbers of beads stuck at the cup stage and aberrant repetitive actin ring condensations during particle engulfment, resembling failed completion of phagosome closure. Overall, in this project we showed that Nir2 and Nir3 proteins regulate phagocytosis by maintaining PI(4,5)P2 levels during particle engulfment at plasma and phagosome membrane. We propose that this is important for actin cytoskeleton remodelling during phagocytic cup formation and phagosome closure, an essential step for successful target engulfment.

Citation (ISO format)
KABA LUNGHI, Mayis. The role of phosphatidylinositol transfer proteins Nir2 and Nir3 during phagocytosis. 2023. doi: 10.13097/archive-ouverte/unige:168707
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Creation05/10/2023 3:15:13 PM
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